Biomolecules, such as deoxyribonucleic acid (DNA) and other nucleic acids, play an important role in cellular functioning as well as control of processes at the tissue, organ, system, and organism level. DNA is a biomolecule formed from two helically-arranged strands; each strand is a biopolymer formed from a linear sequence of four fundamental nucleotide units or bases—adenine, cytosine, guanine and thymine, abbreviated respectively as A, C, G and T. Each base on one strand is hydrogen bonded to exactly one complementary base (A with T and G with C) on the opposite strand, thereby forming a nucleotide base pair. DNA found in a human cell typically consists of approximately three billion base pairs. The specific sequence of A, C, G and T bases which forms the DNA molecule within the cells of an individual is referred to as that individual's genotype, which can vary from person to person. Subsections of the DNA biomolecule may serve to encode for specific proteins; these subsections may consist of several thousands (to millions) of base pairs, and are referred to as genes.
In some cases, a complete determination of the sequence of bases in a DNA molecule—referred to as “DNA sequencing”—defines an individual's genotype, and can be helpful in assessing the propensity for acquiring disease or metabolizing medications, for example. More often, however, knowledge of the sequence within a small subset of the DNA molecule is very useful. For example, confirmation of a specific sequence for a specific gene may be sufficient to assess a predisposition for the development of a certain cancer. The determination of the presence of specific subsection sequences of DNA is referred to as molecular diagnostics; in vitro molecular diagnostics is the term applied when this determination is accomplished using a sample of tissue extracted from an organism.
Molecular diagnostic techniques are typically based on the use of primer molecules, which are short, specific sections of single-stranded DNA used to identify the presence of specific genes or specific sequences within DNA molecules. Primer molecules may, for example, be gene-specific, disease-specific, or organism-specific. Primers and associated reagents are typically mixed with sample of unknown DNA in solution, and the presence of a specific type of DNA is determined through an amplification reaction: if a given DNA sequence is present in the unknown sample, it can be amplified and detected as the reaction proceeds. A widely-used form of amplification is based on the polymerase chain reaction (PCR) technique.
Detection of amplification products (referred to as amplicons) or monitoring of the amplification process itself may be accomplished in numerous ways. One of the most common techniques detects a fluorescence optical signal which increases in intensity during the amplification process if the target DNA sequence is present in an unknown sample. Traditional detection approaches for monitoring the progress of an amplification process use optical sensors (e.g., photodiodes or phototransistors) for sensing the fluorescent light.
The ability of a monitoring system to detect the fluorescence signal ultimately impacts the sensitivity with which low concentrations of DNA can be detected. In many cases the initial quantity of DNA may be extremely small. One problem known in the prior art is the inability to detect very small quantities of DNA due to susceptibility to noise sources inherent in any monitoring system, including optical noise, detector noise, and electrical signal processing noise.
It is desirable to have an apparatus capable of both amplifying nucleic acid under optimal conditions and monitoring the amplification while rejecting undesired signals introduced by sources of noise.
It is desirable to have an apparatus capable of amplifying and monitoring nucleic acid with improved sensitivity for nucleic acid detection.